Eukaryotic cells have evolved complex intracellular organelles which effectively separate biochemical pathways. However, most of the proteins involved in the enzymatic reactions must be imported into the organelles across one or more membranes. A striking example of this transport process occurs with mitochondria, which imports over 90% of its proteins and segregates these proteins to the outer membrane, the intermembrane space, the inner membrane, or the matrix. In this proposal, I outline experiments that may define and locate the targeting and sorting sequences in 1-pyrroline 5-carboxylate (P5C) dehydrogenase (a matrix protein) and cytochrome c peroxidase (an intermembrane space protein). These signal sequences will be analyzed by mutating cloned genes encoding the proteins and subsequently determining the effect of those mutations on protein translocation and segregation in isolated mitochondria. Mutants will be screened using an in vitro transcription-translation-translocation assay that is designed to speed not only the construction and analyses of the mutated genes, but also the analyses of the translocation pathway. Although the initial screening will be done in vitro, interesting translocation mutants will be inserted into appropriate yeast strains and the in vivo location of the mutated protein determined to ensure that the in vitro results faithfully reproduce the in vivo pathway. In addition to characterizing the targeting and sorting sequences, I propose to examine a particular step in the translocation process: that is, how are proteins which are completely folded in the cytoplasm transferred across the mitochondrial membranes? Experiments outlined in this application may determine whether protein translocation occurs by a membrane fusion or vesicle formation process, or by unfolding the protein as it is translocated through membrane lipids or proteins. Probes of the transfer step will be constructed and used both in vitro and in vivo to examine the molecular mechanism involved in the transfer step. These experiments may provide insights into the transport and segregation processes involved in mitochondrial biogenesis.